Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

Ahmad, Patricia M.; Feltman, D S; Ahmad, Fazal

doi:10.1042/bj2030045

Cited by 9 publications

(4 citation statements)

References 28 publications

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“…serine loading sites per dimer, we calculated that the serine sites were 73% and 81% saturated by malonate in enzyme preparations 1 and 2 respectively. The malonate binding in the present experiments, 2.9-3.6mol/mol of enzyme (Table 1), is similar to the values reported for rat liver enzyme by Stern et al (1982) andAhmad et al (1982), 3.0 and 3.5 mol/mol of enzyme respectively. The present values are higher than the values reported for malonate binding to chicken liver enzyme (Kumar, 1982) and rabbit mammary-gland enzyme (McCarthy & Hardie, 1983), 2.0 and 2.2-3.1 mol/mol of enzyme respectively.…”

Section: Resultssupporting

confidence: 90%

Stoichiometry of substrate binding to rat liver fatty acid synthetase

Mikkelsen¹,

Smith²,

Stern³

et al. 1985

Biochemical Journal

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Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

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Section: Resultssupporting

confidence: 90%

Stoichiometry of substrate binding to rat liver fatty acid synthetase

Mikkelsen¹,

Smith²,

Stern³

et al. 1985

Biochemical Journal

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“…Amino acid analysis For amino acid analysis 20-40 ,ug of pyruvate carboxylase was hydrolysed with toluene-p-sulphonic acid containing 0.2% 3-(2-aminoethyl)indole including 3-guanidinoalanine, which served as an internal standard (Liu & Chang, 1971). The hydrolysis was performed in vacuo for 48 h at 110°C, and the hydrolysates were analysed on a Beckman 120 C amino acid analyser (Ahmad et al, 1982). Enzyme assays Pyruvate carboxylase was assayed at 37°C by a 14CO2-fixation assay (Atkin et al, 1979).…”

Section: T3-l Cellsmentioning

confidence: 99%

Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay

Ahmad

Méndez³

1986

Biochemical Journal

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A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.

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“…Materials. Rat mammary gland fatty acid synthase was purified by the method of Ahmad et al (1982). Activity assays were conducted at 30 °C with the procedure of Smith & Abraham (1975).…”

Section: Methodsmentioning

confidence: 99%

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

Miziorko¹,

Behnke²,

Ahmad³

et al. 1986

Biochemistry

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3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)

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Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

Cited by 9 publications

References 28 publications

Stoichiometry of substrate binding to rat liver fatty acid synthetase

Stoichiometry of substrate binding to rat liver fatty acid synthetase

Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

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